1. Field of the Invention
The present invention generally relates to a biological test method and more particularly to an improved method of measuring bacterial endotoxins in blood fractions, particularly human blood fractions, and an improved method of stabilizing king crab amebocyte lysate.
2. Prior Art
Various methods have been devised for the detection of bacterial endotoxins in human and animal blood. One of the newer methods involves the use of Limulus amebocyte lysate. In that method, the lysate is contacted with an endotoxin-containing source such as a human blood fraction which has been previously extracted with chloroform or the like or diluted substantially with water to reduce the concentration of an inhibitor in the blood to below the level which would substantially impair the desired gelation of the lysate by blood endotoxins. The previously diluted or purified blood fraction is mixed with the lysate and the endotoxins in the blood cause the lysate to form a clot.
Unfortunately, test results have varied widely, due to the variable nature of the lysate. However, advances have been recently made in the purification of the lysate to improve the test results. See for example, U.S. Pat. No. 4,107,077 wherein a member of a selected group of organic solvents is utilized to extract inhibitors from the lysate in order to improve the sensitivity of the lysate to blood endotoxins. The firmness and extent of the clot formed by the gelation reaction is measured subjectively in this test by viewing the same, in some cases while inverting the tube containing the clot. Therefore, a true quantitative determination of the concentration of endotoxin cannot be made utilizing this method. Moreover, the method requires skilled personnel, is not always accurate, and takes a considerable length of time to perform, of the order of 45-90 minutes.
Bacterial endotoxins are produced by Gram negative bacteria, many of which are very dangerous or deadly in human beings and animals. Symptoms range from mild to high fever and in many cases death results. It is extremely important in order to promptly initiate the proper medical treatment to identify as soon as possible the fact that endotoxins are present in the blood fraction sample and, if possible, the concentration of the endotoxins. The previously described gelation reaction test method is deficient because of the considerable length of time necessary to carry it out, because it does not accurately measure endotoxin concentration, because it is difficult to standardize and because it requires highly skilled experienced personnel to perform it.
A new chromogenic substrate method for assaying bacterial endotoxins using Limulus amebocyte lysate is described in pages 209-220 of "Biomedical Applications of the Horseshoe Crab" (1979) Allen R. Lis, Inc. That method is specified as not being applicable to blood and blood fractions because of the inhibitors in the blood. Instead, the disclosure is directed to the testing of bacterial solutions containing endotoxins such as might be the case, for example, in testing food for contamination. German Auslegeschrift No. 27 40 323 discloses a similar process to the one described in the above-indicated literature reference. The test specimens utilized in the disclosure in the Auslegeschrift are solutions derived from bacterial sources other than blood. Such procedures have not been utilized in testing for blood endotoxins.
It would therefore be highly desirable to be able to provide an improved method of determining bacterial endotoxins in human and animal blood. Such method should be rapid, reproducible, simple to conduct and inexpensive and should preferably result in a quantitative determination of endotoxin concentration. It would also be highly desirable if the method could employ standardized measuring equipment utilizable by relatively unskilled personnel.
It has also been found that, although lyophilized lysate retains its potency under suitable storage conditions whenever liquid king crab amebocyte lysates are used, whether original or reconstituted from the powder form, they tend to deteriorate in potency rapidly. Thus, their reactivity to bacterial endotoxins sharply and progressively decreases with time before and during tests. Accordingly, variable test results are a common occurrence, especially when utilizing liquid lysates of different ages. It would therefore be highly desirable if a method could be devised to improve the stability of the liquid lysates in order to increase the accuracy of tests involving their use.